Conventionally, fluorescent light has been used to generate microscopic images of histological slices of biological tissue using so-called fluorescence microscopy. However, tissue sectioning used in conventional fluorescence microscopy is limited to slice thicknesses (i.e., tissue depths) on the order of half a millimeter, and therefore, conventional fluorescence microscopy is not appropriate for imaging through entire organs or through the whole human body.
In order to provide images deeper into tissue, conventional systems and techniques have used light sources and fluorochromes that emit near infrared light. The near infrared light is selected because near infrared light has low absorption and can penetrate several centimeters into biological tissue. Near infrared light is used in a variety of optical imaging systems and techniques.
Fluorescent light can be emitted from a tissue in response to an excitation light source transmitting excitation light into the tissue. The excitation light excites the emission of fluorescent light from fluorochromes, some of which are man-made molecules, within the tissue. The excitation light can also excite the emission of fluorescent light from fluorescent proteins within the tissue, including fluorescent proteins that do not naturally occur within tissue. The excitation light can also excite the emission of fluorescent light from naturally occurring molecules within the tissue, generating so-called autofluorescence.
Fluorescence reflectance imaging (FRI) is a conventional technique used to generate microscopic and macroscopic images of fluorescent light emitted by a biological tissue. An FRI system transmits light onto and/or into biological tissue and collects fluorescence light that is emitted back from the tissue. In fluorescence reflectance imaging, excitation light (for example, near-infrared light) from an excitation light source is used to illuminate the tissue. In some existing systems, the excitation light source is used to excite exogenously administered fluorochromes within the tissue that, in turn, emit fluorescent light. Alternatively, in some existing systems the excitation light source is used to excite naturally occurring fluorogenic molecules (i.e., resulting in autofluorescence) within the tissue that emits fluorescent light. The emitted fluorescent light can be visually inspected or it can be captured with a CCD camera or other photon detector positioned generally on the same side of the tissue as the excitation light source.
Fluorescence transillumination imaging (FTI) is another conventional technique used to generate macroscopic images of fluorescent light emitted by a biological tissue. As with FRI, in FTI, excitation light (for example, near infrared light) from an excitation light source is used to illuminate a tissue, and the excitation light propagates into the tissue, exciting the emission of fluorescent light from within the tissue. However, in contrast to the above-described fluorescence reflectance arrangement, in fluorescence transillumination imaging, a CCD camera or other photon detector is positioned generally on the opposite side of the tissue from the excitation light source. In some arrangements, the emitted fluorescent light is near infrared light.
Fluorescence reflectance imaging (FRI) and fluorescence transillumination imaging (FTI) are forms of “planar” imaging, which provide two-dimensional images. More advanced optical imaging systems and methods have been developed, which utilize tomographic methods. These systems and methods operate by obtaining photonic measurements at different projections (i.e., angles) to the tissue and combining the measurements using a tomographic algorithm. Advantages of tomography include an ability for image quantification of deep fluorochromes, and an ability to provide three-dimensional imaging with feature depth measurements. In some applications, tomography has been used in-vivo to measure enzyme regulation and treatment response to drugs. However, tomography is more complex than planar imaging, requiring more advanced instrumentation, requiring multiple illumination points (projections), which can require multiple light sources, and requiring advanced theoretical methods for modeling photon propagation in tissues.
Some fluorescence imaging systems use a catheter-based or an endoscopic arrangement, wherein an excitation light source and a light receiver are coupled via fiber-optic bundle to a catheter-based or endoscopic instrument (hereafter referred to as an insertable instrument), which can be inserted, for example, within a body cavity, organ, or vessel.
The insertable instruments have resolved some of the imaging depth limitations of the above described fluorescence reflectance imaging by allowing intravital access to pathologies deeper inside the body, such as bladder cancer, ovarian cancer, lung cancer, bronchial cancer, and colonic polyps and cancer, as well as arterial plaque. However, with the above-described insertable arrangement, it can be recognized that, as the insertable instrument is moved within the body, the fluorescent light in each image will vary, because the distance from the insertable instrument to the tissue and the angle relative to a surface of the tissue being imaged can vary greatly, making quantifiable assessment of fluorescence difficult from image to image. Variable distances and angles of the insertable instrument during image acquisition result in variable light paths for each image frame, both for excitation light traveling to the tissue and for emission light traveling back to the insertable instrument. Different light paths, in turn, cause different levels of light at an image recording device, and ultimately result in variable signal intensities in each image as images are collected in real time. The variable intensities in each image make it difficult to compare images as they are sequentially captured.
Conventional insertable arrangements are also limited to 8-bit dynamic range for each of two or three optical channels (e.g., a channel to form an image of the excitation light, and one or two channels to form images of fluorescent light).
In general, an intensity of fluorescent light received from a biological tissue is indicative of a concentration of fluorescent molecules within the biological tissue. However, since conventional insertable arrangements have limited ability to provide a quantifiable and correct intensity of images in the various collected images as the insertable instrument is moved about, it is not generally possible in conventional insertable arrangements to determine concentrations of fluorescent light generating molecules in the tissue or to provide an image indicative of the concentrations in real-time.